Ischemic Preconditioning Attenuates Cardiac Sympathetic Nerve Injury via ATP-Sensitive Potassium Channels During Myocardial Ischemia
Background During myocardial ischemia, massive norepinephrine (NE) is released from the cardiac sympathetic nerve terminals, reflecting the sympathetic nerve injury. A brief preceding ischemia can reduce infarct size; this is known as ischemic preconditioning (PC). The effect of PC on sympathetic nerves, however, including its underlying mechanisms in dog hearts, has remained unclear. Thus, this study was designed to elucidate whether the activation of ATP-sensitive potassium (KATP) channels is involved in the mechanism of cardiac sympathetic nerve protection conferred by PC.
Methods and Results Interstitial NE concentration was measured by the in situ cardiac microdialysis method in 45 anesthetized dogs. Five minutes of ischemia followed by 5 minutes of reperfusion was performed as PC. In the controls, the dialysate NE concentration (dNE) increased 15-fold after the 40-minute ischemia. PC decreased dNE at 40-minute ischemia by 59% (P<0.01), which was reversed by glibenclamide. A KATP channel opener, nicorandil (25 μg · kg−1 · min−1 IV), decreased dNE at 40 minutes of ischemia by 76% (P<0.01), which was also reversed by glibenclamide. During the PC procedure, no significant increase in dNE was detected, even with the uptake-1 inhibitor desipramine.
Conclusions Cardiac sympathetic nerve injury during myocardial ischemia was attenuated by PC via the activation of KATP channels, but the trigger of the PC effect is unlikely to be NE release in dog hearts.
Received March 2, 2001; revision received May 1, 2001; accepted May 7, 2001.
A brief episode of myocardial ischemia confers myocardial protection against a following prolonged myocardial ischemia. This effect is known as ischemic preconditioning (PC).1 Several possible triggers for the PC effect were postulated, including adenosine, norepinephrine (NE), and bradykinin.2–7 Protein kinase C can be activated by these triggers and mediates the PC effect.8,9 Because the effect of PC induced by protein kinase C activation is blocked by the ATP-sensitive potassium (KATP) channel blocker, the downstream of protein kinase C could be KATP channel opening.10–12
The previous studies of the effect of PC are focused primarily on the infarct size limitation, but its effect on cardiac sympathetic nerve injury remains unknown. During myocardial ischemia, massive myocardial NE release into the interstitial space was observed, caused by a nonexocytotic mechanism reflecting sympathetic nerve injury.13,14 Recently, the myocardial release of NE after prolonged ischemia was attenuated by a preceding transient ischemia in rat and rabbit hearts,15,16 suggesting the beneficial effect of PC on sympathetic nerve injury. The underlying mechanism of PC for cardiac sympathetic nerve protection, however, remains unknown. Thus, in the present study, interstitial NE concentration during the coronary artery ligation was measured by the cardiac microdialysis method, and the effect of PC on interstitial NE release was evaluated. To elucidate whether the underlying mechanism of PC on sympathetic nerves involves the KATP channel, the effect of KATP channel blocker and its opener was evaluated. Furthermore, we evaluated whether the trigger of PC is intrinsic NE by measuring NE release during PC procedures.
Experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication No. 86-23). The protocol was approved by the Animal Research Committee of Yamaguchi University School of Medicine. Beagle dogs (n=45, 10 to 14 kg) were anesthetized with ketamine (15 mg/kg SC) followed by infusion of α-chloralose (150 mg/kg IV). Anesthesia was maintained by continuous infusion of α-chloralose (25 mg · kg−1 · h−1). Dogs were intubated and ventilated with a Harvard respirator. The arterial Po2, Pco2, and pH were measured and maintained within normal ranges. After a thoracotomy in the fifth intercostal space, the heart was suspended in a pericardial cradle. A high-fidelity catheter-tip micromanometer (Millar Instruments) was inserted into the left ventricle (LV) to measure LV pressure. A pair of ultrasonic dimension gauges (Triton) was placed in the midwall of the territory of the left anterior descending coronary artery (LAD). An occluder was placed around the proximal portion of the LAD. Hemodynamic data were recorded on a digital recording system (CODAS, Data Q). Myocardial segment length shortening, the peak of the first derivative of LV pressure, and the time constant of isovolumic LV pressure decay (τ) were calculated as previously reported.17
Regional Myocardial Blood Flow
Six million colored microspheres (15 μm in diameter, Dye-Trak, Triton) were injected into the left atrium, and the arterial blood was withdrawn at a rate of 6 mL/min for 90 seconds. Trypan blue dye (Sigma Chemical Co) was injected into the coronary artery to determine the risk area. A block of myocardium from risk and nonrisk areas was cut out and divided into endocardial, midmyocardial, and epicardial thirds. After having been weighed, myocardium was dissolved in 16 mol/L KOH. The suspension was filtered (pore size 10 μm, Triton) to collect microspheres, which were dissolved by dimethyl formamide (Wako) to extract color dyes. The color spectra were measured by a spectrophotometer (U-2000, Hitachi), and the myocardial blood flow was calculated as previously described.18
A microdialysis probe (OP-100, EICOM) consisted of a polyacrylonitrile membrane (cutoff molecular weight 50 000 Da). The probe was implanted into the midwall of the myocardium with a guiding needle. Ringer’s solution with low-molecular-weight heparin (dalteparin sodium, 20 U/mL, Kissei) was perfused through the dialysis probe at a rate of 3 μL/min with a microsyringe pump (EP-60, EICOM). The dialysate was collected every 10 minutes with a fraction collector (EF-80, EICOM) cooled to 3°C. Collection tubes contained 20 μL of 0.1-mol/L acetic acid to prevent NE degradation. Dialysates were frozen at −80°C until use. The position of the dialysis probe was checked and proper alignment confirmed at the end of each experiment.
Measurement of Dialysate NE Concentration
The dialysate NE concentration (dNE) was measured by high-performance liquid chromatography.19,20 As an internal standard, 100 pg dihydroxybenzylamine (Sigma) was added to each dialysate sample. The mobile phase consisted of 5% methanol, 1.85 mmol/L sodium 1-octanesulfonate (Nacalai), 0.13 mmol/L disodium EDTA, 113 mmol/L NaH2PO4 · 2H2O, 100 mmol/L Na2HPO4 · 12H2O, and purified water. From the chromatogram, peak areas of NE (A) and dihydroxybenzylamine (B) were calculated, and the dNE (in nmol/L) was obtained by the following equation: [dNE]=A/B×[DHBA]/(recovery rate)×0.33.
The recovery rate in the present system was 20.7±4.2%, which was checked in vitro before each experiment.
After the microdialysis probe had been implanted, ≥60 minutes were allowed to elapse for the equilibration. Dogs were divided into the following 6 groups (n=6 in each group) (Figure 1). In the control group, the LAD was ligated for 40 minutes. In the PC group, a single 5-minute coronary occlusion followed by 5-minute reperfusion as a PC procedure preceded the sustained 40-minute ischemia. In group 3, glibenclamide (0.2 mg/kg bolus followed by 0.06 mg · kg−1 · min−1 IV, Sigma) was administered 10 minutes before the PC procedure and continued throughout the 40-minute ischemia. To eliminate the influence of hypoglycemia caused by glibenclamide, 0.28 mol/L glucose was infused. In group 4, nicorandil (25 μg · kg−1 · min−1 IV, Chugai) was started 20 minutes before and continued throughout the sustained 40-minute ischemia. In group 5, glibenclamide was coadministered with nicorandil in the same manner as in groups 3 and 4. In group 6, diltiazem (20 μg · kg−1 · min−1 IV, Tanabe), was infused 20 minutes before and continued throughout the 40-minute ischemia. To examine the effect of myocardial ischemia and glibenclamide infusion on the NE concentrations in the systemic blood, systemic venous blood was sampled before and after the glibenclamide infusion and after myocardial ischemia had been created (n=4). In another series of experiments (n=5), the change in dNE during the PC procedure was evaluated with and without an uptake-1 blocker, desipramine (1 mg/kg IV, Sigma).
All values are expressed as mean±SEM. The serial changes of hemodynamics and NE concentrations were assessed by a repeated-measures ANOVA, followed by Scheffé’s multiple comparison test. Comparisons among groups were tested by 1-way ANOVA followed by Scheffé’s multiple comparison test. A value of P<0.05 was considered statistically significant.
Hemodynamics are shown in Table 1. Compared with the preischemic state, segment length shortening decreased (P<0.05) and τ prolonged (P<0.05) in all groups after the 40-minute ischemia. Heart rate, peak LV pressure, and peak +dP/dt did not change after the 40-minute ischemia in all groups. All parameters did not differ significantly among 6 groups.
Regional Myocardial Blood Flow
Table 2 shows the myocardial blood flow of the endocardial third in both ischemic and nonischemic regions. Before the coronary occlusion, myocardial blood flow was significantly higher in the nicorandil group than the control group (P<0.05). After the 40-minute ischemia, the myocardial blood flow in the ischemic region became <0.15 mL · min−1 · g−1 in all groups, and no significant differences were noted among the groups.
dNE During Prolonged Ischemia
Figure 2 shows changes in dNE during myocardial ischemia. In the control group, after 40 minutes of ischemia, dNE increased 15-fold of the preischemic level. With PC, dNE was attenuated significantly, by 59%, compared with that of the control group (P<0.01 versus control). The effect of PC was abolished by pretreatment with glibenclamide. Indeed, dNE was more greatly increased by pretreatment with glibenclamide than in the control group (103.3±22.3 versus 64.6±19.5 nmol/L, P<0.05). Nicorandil attenuated dNE by 76% after 40 minutes of ischemia compared with that of the control group (P<0.01 versus control). The effect was also abolished by glibenclamide. Surprisingly, dNE concentration was further increased by pretreatment with glibenclamide compared with the control group (126.8±24.9 nmol/L, P<0.05). In the diltiazem group, no significant attenuation in dNE was observed during the 40-minute ischemia compared with the control group.
NE Concentration in Systemic Venous Blood
Figure 3 shows serial change in NE concentration in the systemic venous blood. Without ischemia, glibenclamide administration itself did not change the systemic venous NE concentration. During the 40-minute ischemia, the NE concentration in the systemic venous blood did not change significantly even in the presence of glibenclamide.
dNE During PC Procedures
Figure 4 shows changes in dNE before and during PC procedures. Without desipramine, the PC procedure did not increase dNE. With desipramine, the basal dNE increased ≈3-fold, but the PC procedure did not further increase the dNE.
The major findings of the present study are that (1) during prolonged myocardial ischemia, marked elevation of dNE was observed without a concomitant increase in the NE level in the systemic blood; (2) the elevation of dNE during the 40-minute ischemia was markedly attenuated by PC and by a KATP channel opener, nicorandil; (3) the involvement of KATP channels in the effect of PC on sympathetic nerves was confirmed, because the effect was inhibited by glibenclamide; and (4) the dNE during the PC procedure was not increased, suggesting that the trigger of PC is unlikely to be the intrinsic NE release. This study is the first that demonstrates the underlying mechanism of the sympathetic nerve protection conferred by PC.
In Vivo Microdialysis
We demonstrated a 15-fold increase in dNE during 40-minute ischemia by microdialysis. Measurement of dNE by cardiac microdialysis was first reported by Akiyama et al21 and has since been shown to be reliable. We confirmed the reliability of this method by measuring the exocytotic release of NE under desipramine (Figure 4). Desipramine is an uptake-1 inhibitor blocking the presynaptic reuptake of NE. Thus, the basal dNE should increase under desipramine. As expected, dNE was increased 3-fold by desipramine.
dNE During Myocardial Ischemia
The dNE during coronary artery occlusion should reflect the NE release from the sympathetic nerve terminals of the ischemic myocardium. The massive release of NE from the sympathetic nerve terminals can be attributed to nonexocytotic release, because the exocytotic release of NE is inhibited during myocardial ischemia.13,14 dNE during coronary artery occlusion could be influenced by the washout, which is dependent on the collateral circulation. Because the collateral blood flow measured in the present study was not significantly different among the groups, the washout rate is not a major factor influencing the changes in dNE.
Influence of NE in Systemic Blood
Another factor influencing the dNE is the NE level of the systemic blood. If the NE level of the systemic blood changes, the dNE could be influenced through the collateral blood flow. As shown in Figure 3, no significant changes in NE concentrations in the systemic blood were observed before or during the 40-minute ischemia. This indicates that the systemic blood has negligible influence on the changes in the dNE. Thus, the observed changes in dNE in the present study are attributed primarily to the changes in the release from the sympathetic nerve terminals of the myocardium.
Effect of PC on dNE During Prolonged Ischemia
We demonstrated that ischemia-induced NE release was attenuated by PC, indicating the protective effect of PC on ischemic sympathetic nerve injury. A similar finding was reported by Seyfarth et al15 in isolated perfused rat hearts. The underlying mechanism of the neural protection, however, has until now remained unknown. The present study demonstrates for the first time that the opening of KATP channels plays a pivotal role in the mechanism of PC for neural protection in the dog heart.
Effect of KATP Channel Opener on dNE
We demonstrated that a KATP channel opener, nicorandil, attenuated ischemia-induced release of NE and that its effect was abolished by glibenclamide. This finding strengthens the hypothesis that the opening of KATP channels is neuroprotective. Because the existence of KATP channels on nerves has been demonstrated22,23 and their physiological function is likely to modulate the release of NE,24 it is conceivable that KATP channels on the sympathetic nerve terminals act as an effector of neural protection. It has been shown that the administration of high-dose KATP channel openers causes abbreviation of the action potential duration, which is proarrhythmic. The effects of nicorandil on sympathetic nerve terminals, however, were without any proarrhythmic effects in the present study. This may be explained by the findings that the affinity of nicorandil for KATP channels of nerves is higher than that of smooth muscles or myocytes.23 Thus, a lower dose of nicorandil may be sufficient for protecting nerve terminals than for myocytes.
As shown by Schömig et al,13,14 ischemia-induced NE release is mediated by starvation of axoplasmic ATP, leading to counterdirectional NE release through uptake-1 carriers on the sympathetic nerve terminals. This process is dependent on intracellular Na+, but not on Ca2+.13,14 Therefore, the mechanism of KATP channel activation for reducing the ischemia-induced NE release may involve the inhibition of intracellular Na+ accumulation as well as the preservation of axoplasmic ATP.
Excessive Response of dNE Under Glibenclamide
Without ischemia, we showed that glibenclamide itself did not induce systemic NE release, as shown in Figure 3. We also confirmed that the dNE was not increased by the infusion of glibenclamide without ischemia (data not shown). To our surprise, however, glibenclamide intensified the increase in dNE during myocardial ischemia even with PC or nicorandil. The difference of washout rate can be excluded, because the collateral blood flow was not changed by glibenclamide. The reuptake rate should not influence the result, because the reuptake is inhibited during myocardial ischemia. A possible explanation for the phenomenon is that the intrinsic opening of the KATP channels, which seemingly protects the cardiac sympathetic nerves, is blocked by glibenclamide. This is in contrast to a previous report that glibenclamide did not increase infarct size more than control.12 This discrepancy may be caused by the different vulnerabilities between myocytes and sympathetic nerves.
Effect of Calcium Antagonist on dNE
No significant effect on dNE during myocardial ischemia was observed with the calcium channel blocker diltiazem at the dose used. This finding supports the idea that the neuroprotection is specific for KATP channels at this dose that the systemic hemodynamics does not change. If the dose of diltiazem increased, however, the protective effect may be observed, accompanied by a reduction of oxygen consumption.25
dNE Change During PC Procedures
During PC procedures, no significant changes in dNE were observed (Figure 4), indicating that exocytotic NE release is negligible in the dog heart. This was further confirmed in the presence of the uptake-1 inhibitor desipramine, which inhibits the reuptake of NE. The finding that the PC procedure did not elevate dNE even in the presence of desipramine strongly suggests that NE is not a trigger of PC in the dog heart. The activation of α1-adrenergic receptors mimics PC through protein kinase C activation.4,5 This pathway, however, would not have been activated in our model. Thus, other triggers, such as adenosine, would be an important trigger of PC for sympathetic nerve protection in the dog heart. Because species differences exist in the trigger of PC for infarct size–limiting effect,26–29 it is reasonable that the trigger of PC for neural protection would be different in other species, such as rats and rabbits.
Excessive stimulation by NE during myocardial ischemia is deleterious to the ischemic myocardium by inducing intracellular calcium overload and the degradation of cytoskeletal structures, leading to expansion of the infarct size and stunned myocardium.30,31 Furthermore, reperfusion arrhythmias are related, in part, to excessive NE release during myocardial ischemia.32 Recently, the excessive stimulation of NE was shown to be cardiotoxic because of the instability of Ca2+-releasing channels caused by the phosphorylation of FKBP12.6 through protein kinase A.33 Considering this evidence, the cardiotoxicity caused by the exposure of the ischemic myocardium to high concentrations of NE could be attenuated by PC or KATP channel openers.
A recent clinical study indicated that injury of the cardiac sympathetic nerves detected by [123I]meta-iodobenzylguanidine (MIBG) imaging exceeded the area of necrosis. This indicates that the vulnerability of sympathetic nerves and myocytes to ischemia may be different.34 Because sympathetic nerve dysfunction causes critical arrhythmias,35 it is intriguing to study whether the protective effect of KATP channel opening on sympathetic nerves contributes to the inhibition of critical arrhythmias after acute myocardial infarction.
This work was supported in part by Grants-in-Aid for Scientific Research 0767 and 0785 from the Ministry of Education, Science, and Culture of Japan.
Murry CE, Jennings RB, Reimer KE. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation. 1986; 74: 1124–1136.
Liu GS, Thornton JD, Van Winkle DM, et al. Protection against infarction afforded by preconditioning is mediated by A1 adenosine receptors in rabbit heart. Circulation. 1991; 84: 350–356.
Downey JM, Liu GS, Thornton JD. Adenosine and the anti-infarct effects of preconditioning. Cardiovasc Res. 1993; 27: 3–8.
Bankwala Z, Hale SL, Kloner RA. α-Adrenoceptor stimulation with exogenous norepinephrine or release of endogenous catecholamines mimics ischemic preconditioning. Circulation. 1994; 90: 1023–1028.
Banerjee A, Locke-Winter C, Rogers KB, et al. Preconditioning against myocardial dysfunction after ischemia and reperfusion by an α1-adrenergic mechanism. Circ Res. 1993; 73: 656–670.
Goto M, Liu Y, Yang XM, et al. Role of bradykinin in protection of ischemic preconditioning in rabbit hearts. Circ Res. 1995; 77: 611–621.
Ytrehus K, Liu Y, Downey JM. Preconditioning protects ischemic rabbit heart by protein kinase C activation. Am J Physiol. 1994; 266: H1145–H1152.
Schulz R, Rose J, Heusch G. Involvement of activation of ATP-dependent potassium channels in ischemic preconditioning in swine. Am J Physiol. 1994; 267: H1341–H1352.
Toombs CF, Moore TL, Shebuski RJ. Limitation of infarct size in the rabbit by ischaemic preconditioning is reversible with glibenclamide. Cardiovasc Res. 1993; 27: 617–622.
Gross GJ, Auchampach JA. Blockade of ATP-sensitive potassium channels prevents myocardial preconditioning in dogs. Circ Res. 1992; 70: 223–233.
Schömig A, Fischer S, Kurz T, et al. Nonexocytotic release of endogenous noradrenaline in the ischemic and anoxic rat heart: mechanism and metabolic requirements. Circ Res. 1987; 60: 194–205.
Schömig A, Dart AM, Dietz R, et al. Release of endogenous catecholamine in the ischemic myocardium of the rat, A: locally mediated release. Circ Res. 1984; 55: 689–701.
Seyfarth M, Richardt G, Mizsnyak A, et al. Transient ischemia reduces norepinephrine release during sustained ischemia: neural preconditioning in isolated rat heart. Circ Res. 1996; 78: 573–580.
Miura T, Miyazaki S, Guth BD, et al. Influence of the force-frequency relation on left ventricular function during exercise in conscious dogs. Circulation. 1992; 86: 563–571.
Kowallik P, Schultz R, Guth BD, et al. Measurement of regional myocardial blood flow with multiple colored microspheres. Circulation. 1991; 83: 974–982.
Akiyama T, Yamazaki T, Ninomiya I. In vivo monitoring of myocardial interstitial norepinephrine by dialysis technique. Am J Physiol. 1991; 261: H1643–H1647.
Antomarchi HS, Amoroso S, Fosset M, et al. K+ channel openers activate brain sulfonylurea-sensitive K+ channels and block neurosecretion. Proc Natl Acad Sci U S A. 1990; 87: 3489–3492.
Ohe K, Sperlagh B, Santha E, et al. Modulation of norepinephrine release by ATP-dependent K+ channel activators and inhibitors in human isolated right atrium. Cardiovasc Res. 1999; 43: 125–134.
Mizumura T, Auchampach JA, Linden J, et al. PD 81,723, an allosteric enhancer of the A1 adenosine receptor, lowers the threshold for ischemic preconditioning in dogs. Circ Res. 1996; 79: 415–423.
Li Y, Kloner RA. The cardioprotective effects of ischemic preconditioning are not mediated by adenosine receptors in rat heart. Circulation. 1993; 87: 1642–1648.
Sato H, Hori M, Kitakaze M, et al. Reperfusion after brief ischemia disrupts the microtubule network in canine hearts. Circ Res. 1993; 72: 361–375.
Penny WJ. The deleterious effects of myocardial catecholamines on cellular electrophysiology and arrhythmias during ischemia and reperfusion. Eur Heart J. 1984; 5: 960–973.
Matsunari I, Schricke U, Bengel FM, et al. Extent of cardiac sympathetic neuronal damage is determined by the area of ischemia in patients with acute coronary syndromes. Circulation. 2000; 101: 2579–2585.